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JOURNAL OF VIROLOGY, Oct. 1977, p.211-221 Copyright© 1977 American Society forMicrobiology

Vol. 24, No. 1 Printedin U.S.A.

Specific Binding Sites for a Parvovirus, Minute Virus of Mice,

on

Cultured Mouse Cells

P. LINSER,* HELEN BRUNING, AND R. W. ARMENTROUT

Department ofBiological Chemistry, University of Cincinnati Medical Center, Cincinnati, Ohio 45267 Received forpublication 30March1977

The early

interactions between parvoviruses

and host cells have

not been

extensively described previously.

In

this

study

we have characterized some

aspects

of

viral

binding

to

the cell

surface

and demonstrated the existence of

specific cellular

receptor

sites for minute virus

of mice (MVM) on two murine

cell lines that

are permissive

for viral growth.

The interaction had a pH

optimum

of

7.0 to7.2,

and both the

rate

and

extent

of the reactions

wereslightly

affected by

temperature. Mouse A-9 cells (L-cell derivative) had -5 x 105

specific

MVM

binding

sites per

cell,

and Friend erythroleukemia cells had

1.5 x 105

MVM

sites per cell. In contrast, the

nonpermissive

mouse

lymphoid cell

line

L1210

lacked specific viral

receptors.

Also,

cloned lines of A-9

cells resistant

to

viral infection have been isolated. One

of these lines

lacked the

"specific"

virus

attachment sites but exhibited low levels of nonsaturable

virus

binding.

Based

on

these examples, infectivity

is

correlated with the

presence

of

specific viral

receptors on

the cell surface.

We

have looked

atthe initial interaction

be-tween

minute

virus

of

mice

(MVM)

and several

murine tissue

culture

cell

lines

in

order

to

char-acterize

and quantify

a

specific

virus-binding

site. MVM

is a parvovirus, a

class of small

viruses that contain a linear single-stranded

DNA

genome.Very little information concern-ing early parvovirus host cell interactions has

been

previously reported

(19).

However,

the

binding of

picornaviruses to

cells has

been

ex-tensively studied and

is in some respects

analo-gous to

the

parvovirus system. Parvoviruses

and

picornaviruses

are roughly the same size

(-17 to 30 nm in

diameter), and they both

consist

of

an

icosahedral

protein

capsid

sur-rounding

a

single-stranded nucleic acid

genome

(RNA

in

the

case

of

picornaviruses).

The initial

interaction

of

picornaviruses such as poliovirus

with

susceptible host cells has been

character-ized. The

number of specific binding sites per

cell is known (-104/cell)

(10, 14, 15),

and

the

presence

of

specific cellular binding

sites in part

explains

the

cytological specificity of

polio-virus infections. There

isevidence that

parvovi-ruses may be tissuespecific as well (3, 12), and

this

report is the first evidence for a

relation-ship

between

infectivity

and the presence of

specific virus-binding

sites on the host cell.

MATERIALS AND METHODS

Virus stocks. All cell lines were free of

myco-plasmas determined periodically by

autoradiogra-phy. Plaque-purified MVM wasthegenerousgiftof 211

Peter Tattersall. MVM labeled in its DNA with

[methyl-3H]thymidine wasgrowninRT-7cells (pre-viously described[18]). Randomlygrowinginfected cells were labeled for 48 h during the interval of maximumviral encapsulation. Half-confluent mon-olayers of RT-7 cells in Corning T-flasks were in-fected with MVMpurifiedfrom the110Sregion ofa

sucrosegradient. The virus was adsorbedtocells in phosphate-buffered saline(PBS) at37°C for2h.The monolayers were then fed withfresh F-11(minimal

essential medium, Grand Island Biological Co.

[GIBCOQ)

with5%heat-inactivated fetal calfserum

and 100U ofpenicillin and100 ,ug ofstreptomycin per ml.Twenty-fourhours after infection the mono-layers weresubcultured and refed with fresh

me-dium containing25,uCi of[methyl-3Hlthymidineper ml. Forty-eight hours later the monolayers were harvested into 0.01 M Tris buffer (pH 9.0),disrupted

by sonic treatment, andextractedtwicewith5 vol-umesof Freon. The aqueousphasewasbroughtto a

final concentration of 10 mM

MgCl2.

DNase

(Worth-ingtonBiochemicals Corp.) wasadded to0.1mg/ml, and themixture wasincubatedina37°Cwaterbath for60min.Thecrude preparationwasthendialyzed

at4°Covernightagainst3x 1-literchangesof0.01M Tris-0.005 M EDTA (pH 9.0) buffer. The preparation was thenlayered onto 37-ml continuous 15 to 30%

sucrosegradientsandcentrifugedinanSW-27 rotor at25,000 rpm and4°Cfor 6.5 h. The110Speakoffull

virus was located by a combination of the optical density at 260nm,thehemagglutinin activity, and the DNase-resistant, acid-insoluble radioactivity assayed across the gradientaspreviously described (18). The concentration of virus particlesinthe virus preparationswasmeasuredby the opticaldensityat 280 nm and calculated from an El% of71.2 as de-scribed by Tattersall et al. (21). Essentially the

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sameresults were obtained when the -concentration ofparticles wasestimated from the protein concen-tration as determined by fluorescence (5) and using a value of 4 x 106 daltons of protein per mol of particles. Infectivity of preparations of this type is typically 1 infectious unit per 4 x 102 to 6 x 102 particles, as measured by 50% tissue culture infec-tivedose or plaque assay.

A-9 cells, a suspension culture derivative of mouse L-cells (13), were grown in F-11 (minimal essential medium, GIBCO)supplemented with 10% heat-inactivated fetal calf serum and 100 U of peni-cillin and 100 ,ug of streptomycin per ml in suspen-sion atdensities from 5 x 104 to 5 x 105/ml. These cells are permissive for viral growth (20) and repre-sentthe model system used to define several param-etersof virus-cell binding in this study.

Murine Friend-745 erythroleukemia cells (Friend virus-transformed spleen cells) (6, 8) were provided by K. Lowenhaupt. These cells are permissive for viral growth (16a) and can be induced to un-dergo erythropoietic "differentiation" in culture

bydimethyl sulfoxide (Me2SO)treatment (6). These cells are suspension adapted and were grown in Ham F-12 (GIBCO) supplemented with 10% fetal calf serum at suspensiondensities of2 x 104to4 x

105/ml. These cellswere inducedby the addition of 1.8%Me2SO to 4 x 104 cells per mlin fresh F-12. The course ofinduction was monitored by scoring the percentage of cells that were positive for hemoglobin by the benzidine staining technique (6).

The mouse lymphoid cell line L1210 was the gift of J. McCormick. These cells do not support MVM growth (16a) andconstitute the negative control for virus binding used in this study. L1210 cells are also grown in suspension in RPMI 1640 (GIBCO) supplemented with 10% heat-inactivated fetal calf

serumatdensities of 2 x 104 to 5 x 105/ml. Binding assay. Binding ofradiolabeled virus to cells was performed in suspension with periodic gentle mixing to keep cells suspended. Reactions werecarried out in sterile Corning disposable poly-styrene conical centrifuge tubes, as described for each experimentinResults. Unless otherwise

speci-fied,for eachdata point a known quantity of virus in

asmall volume (i.e.,5to 50 ul) was addedto 2 x 105

cells in1ml of buffer. The virus wasstored at-200C

in the sucrose solution of the isolation gradient, conditions which prevented viral aggregation. After the appropriate incubation time, the sample was filtered through a 25-mm Nuclepore filter with 5.0-,um pore size, and the filter, which retained the cells,was washed twice with 20 ml of ice-cold buffer. The filter was then air-dried, solubilized with Soluene-100(Packard), and counted by scintillation spectrophotometry in toluene-based scintillation cocktail. Specific activityof viruspreparations was determined byprecipitation of a sample of virus in

10% trichloroacetic acid on a Whatman GFA glass filter,digestedwithSoluene, and counted by scintil-lation. When radiolabeled virus particles were di-rectlyfiltered, less than 0.1% of the input radioac-tivity was retainedby the filter. The DNase lability oftrichloroaceticacid-precipitable counts in the fro-zer. viruspreparations was negligible.

Selection of resistant cells. A-9 cells resistantto

MVMinfection wereselected by allowingsurviving cells from an infectiontogrowoutandcloningcells from the resultant cell population. A-9 cells were

grown inmonolayer in F-11(minimalessential

me-dium, GIBCO) supplemented with 5% heat-inacti-vated fetal calf serum and 100 U ofpenicillinand 100 ,g of streptomycin per ml. The monolayers were infected as described for RT-7 cell infection (18). Afterlysis of most cells due to infection, a few survi-vors were noted and allowed to repopulate the origi-nal culture vessel over a period of 3 to 4 weeks. After regrowth of the monolayer, the culture now en-riched forcells resistant to MVMinfection was tryp-sinized with 0.25% trypsin in PBS (GIBCO) and diluted to 1 cell per 10 ul and seeded into microwell cloning plates at 10

sgl/well.

During outgrowth of the clones, each culture was monitored for the produc-tion ofviral proteins by hemagglutination assay, and only those cultures free of viral protein were subsequently tested for susceptibility to infection using a 50% tissue culture infective dose assay. Clones were also screened for virus binding using the assay systems described in Results.

Electron microscopy. Normal A-9 cell monolay-ers in 60-mmculture dishes were rinsed in PBS at 4°C, and 106 110S MVM particles per cell were added in a total volume of 1 ml of PBS and allowed to adsorb at 4°C for 2 h. The monolayers were then washed in ice-cold PBS several times, fixed in 3% glutaraldehyde in phosphate buffer for 1 h at 4°C, and postfixed in 1% osmic acid. The monolayers wereembedded in Luft Epon mixture and sectioned with aDuPont diamond knife perpendicular to the plane of growth, as described by Anderson et al. (1). Electron micrographs were taken on a JEOL JEM 100B at 60-kV acceleration voltage.

RESULTS

Several conditions of thevirus-binding

reac-tion were examinedto optimizethe conditions

ofthe assay. Itwas

important

tominimize

up-take of virus into cells. Asuptake ofparticles by cells canbereducedby workingat low tem-peratures, the effectoftemperature onthe vi-rus-binding interactionswasexamined.InFig. 1, 105 virus

particles

percellwereadded to 2.2

X 105A-9 cells in 1 ml of PBS at either4or21°C,

and the mixture was allowed to incubate for30

s or1, 3, 5, 15, 30, 60,or 120min. At the endof theincubationtime,thesamplewas filtered as

described

in Materials and Methods and

washed with ice-cold PBS, and the cell-bound counts per minute were measured. At both 4

and 21°C the reaction appears to consist of a

rapid

component within the first 30min and a

slowcomponent that continuesfor at least 2h.

Adsorption times of upto 4 hhavebeen

exam-ined at 4°C, and the slow reaction appears to

continue

indefinitely

(not

shown).

As tempera-ture appearstohaveonlyaslighteffect onthe

J. VIROL.

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BINDING SITES FOR MVM 213 201

9)

0

E9 E a.

9.

0

50

10

[image:3.504.272.417.260.443.2]

30 60 90 120

Minutes

FIG. 1. Effect oftemperature on the binding of

MVMtoA-9 cells in suspension. A total of2 x105

cells in PBS, pH 7.2, were reacted with 104 [3H]thymidine-labeled 11OS MVMpercell for30sto

2hateither 21 or40C. The reactionwasstopped by filtration of the cell suspension through 25-mm Nu-clepore filters witha

5.0-Ium

pore size. Cell-associ-ated tritiumcountsperminuteareplottedversusthe time. Temperatureaffects therateof thereactionand the shape of the curve only slightly. Symbols: (0) 4CC; (0)21 C.

extentof virus binding, subsequent binding

ex-perimentswereperformedat 40C for 2 hasthe

standard conditions.

Little ifanyof the virus boundtocellsat40C

appears tobe takenupduringthe 2-h

incuba-tion period,as78% of the cell-associatedcounts

can be removed by a brief wash (5

min)

in

Ca2+,Mg2+-free PBS with 0.001 M EDTA(Table 1). Treatment of this sort has no observable

effects on plating efficiency of the cells. Thus,

the bindingassay appearsto measure

primar-ily surface attachment and isnotsignificantly complicated by uptake into cells. This

conclu-sion is supported by the fact that virus previ-ously boundtothe cellsurfacecan be competi-tively displaced by subsequently added unla-beled virus (Fig. 2). In this experiment a sub-saturating amount of labeled virus (104

parti-clespercell) wasallowedtobindtocellsat 40C

for2 h. Increasing concentrations of unlabeled

virus were then added to the cell suspension.

After1h, theamountof residual label boundto

the cellswasmeasured. ItcanbeseenfromFig.

2, curve

B,

that theamountof cell-bound label remains constant until the input multiplicity exceeds 5 x 105 particles per cell. However, once this saturation level has been exceeded,

the additional unlabeled viruseffectively

com-petes with thelabeled virus for attachment to

the cell surface. These results indicate that binding of virustothe cellsurface ismostlikely

areversible reaction and thatamajor portionof

the virus isprobably bound atthe cell surface under theconditions of the bindingassay.

Acritical question in measuringanybinding

TABLE 1. Effect of wash treatment on amount of virus bound tocellsa

cpmbound

Treatment Avg % oftotal

Sample 1 Sample 2

Control 1,248 1,186 1,217 100

EDTA wash 219 334 276 22

a Four samples each containing 2 x 105 cells

sus-pended in1ml ofPBS at40Cwerereacted with 105

[3H]thymidine-labeled MVMparticles per cell for 2 h. At the end of the incubation time the samples were filtered and washed as described in the text. Two samples were then washed for 5 minutes (slow filtration) inCa2OMg2+-freePBS with 1 mM EDTA. A total of 78% of the cell-associated counts was washed off by this treatment.

15

0

N 10 \

0

0~~~~~\

tA

101lo10"

lo12

Io13

(unlabeled]

FIG. 2. Curve of competition between binding la-beled and unlala-beledMVM;A-9 cells suspended in PBS at 40C were reacted with a

fixed

quantity of

[3H]thymidine-labeled MVM (2.4 x109particles).In the first case (curve A, 0) the labeled virus was mixed with increasing amounts of unlabeled virus prior toadsorption to cells (2 x 105). Inthe second instance(curve B, 0)thelabeled virus was allowed to adsorb tothecells(1.7 x 105)for2h, and increasing amounts ofunlabeled virus were subsequently al-lowed to incubate with the cells for 1 h prior to

filtration.The arrow indicates the point at which the input multiplicity reached 5 x105 virusparticles per cell.

to cellsurfaces is the extent to which the reac-tion is specific. Generally, specific attachment is limited and therefore saturates as the amount of input

material

increases. In

addi-tion, attachment of labeled particles to specific sites on the cell surface can be competed by unlabeled particles, and this is shown for MVM

binding

inFig. 2, curve A. In this case a fixed

amountoflabeled virus was added to the cells

along

with increasing amounts of unlabeled

VOL. 24, 1977

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particles. It can be seen that the amount of

bound radioactivity beginstodecline when the total input virus approaches5 x 105input

parti-cles percell. Dueto the very high amountsof

unlabeled virus required, we are unable to

demonstrate complete competition. However, the results indicate thatgreaterthan 75% of the viral binding can be competed by unlabeled

virusand is specific by this criteria.

It has been previously noted that parvovirus binding to erythrocytes (hemagglutination) and cellular debris could be minimizedathigh pH (9).Consequently, the dependence uponpH

of MVMbindingto A-9 cellswasexamined by

usingaspectrumof organic bufferstocoverthe

pH range from 6.5 to 9.0 (Table 2). Figure 3 shows that the binding reaction has an opti-mum pH occurring near neutrality. These

ex-periments were repeated using inorganic

buffers(datanotshown), and the pH optimum

wasbetween 7.0 and 7.2 for the binding

reac-tion.

A second important indication of specific binding of virustocellularreceptors is the

cor-relation of infectivity with virus binding. To further demonstrate thatwe are measuring a

specific binding ofvirus to cells, we isolated a

series of A-9 cell clones that wereresistant to

virusinfection and tested them for the loss of virusbinding.

Resistance A-9 cells.Resistanceof A-9clones

to MVM infection was measured by using a

modified 50% tissue culture infective doseassay

for the production of viral hemagglutinin. Ta-ble 3 shows the data forcomparisonof infection

of control A-9 cells and the clonal derivative

designated 8-E. The cellswere infectedby ad-TABLE 2. Buffer solutions usedtogenerateFig.3a

Concn

Buffer" pK. used pHused

(mM)

BIS Tris 6.46 20 6.50

PIPES 6.80 10 6.75;7.00

BES 7.15 20 7.25

TES 7.50 20 7.50

HEPES 7.55 20 7.75

HEPPS (EPPS) 8.00 20 8.00

Tricine 8.15 20 8.25

Bicine 8.35 20 8.50

Tris 8.30 20 8.75; 9.00

aEach buffer solution also contained0.9%NaCl,

7 x 10-4M CaCl2,and 5 x 10-4MMgC12.

bBIS, N,N-methylenebisacrylamide; PIPES,

pi-perazine-N,N'-bis(2-ethanesulfonic acid); TES, N-tris(hydroxymethyl)methyl-2-aminomethane-

sul-fonicacid; HEPES, N-2-hydroxyethyl piperazine-N'-2-ethanesulfonic acid; BES, N,N-bis(2-hydroxy-ethyl)-2-aminoethane sulfonic acid; HEPPS, N-2-hydroxyethylpiperazinepropanesulfonic acid.

'5

-0

E 10

-5

-6.5 7.0 7.5 8.0 8.5 9.0 pH

FIG. 3. pH optimum for MVM binding reaction; 2 x105 A-9 cells suspended in various buffer combina-tions (listed in Table 2) were reacted with 105 [3H]thymidine-labeled viral particles per cell at40C for2h. The suspensions werefilteredasdescribed in the text, and the cell-associated radioactivity was determined. There is pH optimum at pH 7.0. Each point is the average ofthree determinationsplusor

minus therange.

sorption of dilutions of 110S

MVMstock in PBS

at

370C

for

2h. The control A-9 cells

produce

the

expected dilution-dependent

curve in terms of

hemagglutination activity (Fig. 4). By compari-son, it appears

that the

8-E

cells require

a

5-log-unit-higher

concentration of input virus than

do the control

A-9

cells

to

produce the

same

amount

of

hemagglutinin. The

50% tissue cul-ture infective

dose

curve

for

the

normal

cells

peaks

in the

middle, demonstrating

the cell

growth

dependence

of

viral

production.

At the

highest multiplicity of infection

a

larger

per-centage

of

cells

is

initially infected and

conse-quently inhibited from further cell division.

Cells infected

at a

lower

multiplicity of

infec-tion

can go

through several cell

growth cycles

prior

to

spread of the infection

to

all of

the cells.

The infection

was

allowed

to

proceed for

4

days

to

confluency, explaining the lower

titers

of

viral

protein at the

lowest multiplicities of

in-fection.

In

Fig. 5 virus-binding saturation curves

generated

in

parallel

onnormal A-9and

resist-ant 8-E cells are shown. The A-9 curve is bi-phasic, consisting of a specific,

saturable

com-ponent saturating at

approximately

5 x 105 virus particles bound per celland an apparently nonspecific insaturable component. Virus bind-ing tothe 8-E cells is monophasic and

insatura-ble under this

condition, mimicking

the

non-specific

portion of the A-9

virus-binding

curve.

The 8-E cells appear to have lost the specific virusreceptor present in 5 x 105copies per A-9 cell.

Figure 6 is a saturation curve comparing the

permissive

A-9 cell line and the

nonpermissive

L1210 line. In the case of the permissive A-9

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BINDING SITES FOR MVM 215 TABLE 3. Hemagglutination activityproduced in a tissue culture infectivity assay run on A-9 and 8-E cells in

parallels

Dilution of infectious Hemagglutinin production

stock A-9 Mean 8-E Mean

10-2 64; 64; 128 85 2;4;4 3.3

10-3 128; 128; 256 170 0; 0; 0 0

10-4 128; 512; 512 384 0; 0; 0 0

10-5 64; 64; 64 64 0; 0; 0 0

10-6 32; 8; 8 16 0; 0; 0 0

10-7 2;2;2 2 0; 0;0 0

aA total of 105 cells were seeded into 35-mm culture dishes. Dilutions of stock MVM were adsorbed in 0.5

ml of PBS at

370C

for 2 h. The cultureswere subsequently fed with growth media (see text) and refed daily. Four days after infection, mock-infected control cultures of both cell types had reached confluence. The lowest three dilution samples exhibited gross cytopathology in the A-9 cultures, whereas no growth inhibition or cytopathic effect was evident in any 8-E cultures. At this time cells wereharvested into 0.01 M Tris-0.005 M EDTA (pH 9.0) buffer andlysed by sonic treatment. Viral protein was assayed by thestandard hemagglutination assay.

5

cam 4

x 3

2 .

in

I0

x

[image:5.504.50.448.97.189.2]

.i

dilution

FIG. 4. Tissue culture infectivityassayperformed oncontrol A-9 cells andaresistant cloneofA-9 cells

designated 8-E: 105 A-9 cells or8-E cells in

mono-layerwere infected with the dilutions ofstock virus

shownby adsorptioninPBSat370Cfor2 h.After4

days in growth media all ofthe 8-E cultures and

severalofthe A-9 cultures had reachedconfluency.

Atthistime, each samplewasharvestedinto 0.01 M

Tris-O.005 M EDTA (pH 9.0) buffer, sonically treated,andassayed forviralprotein bythe hemag-glutinationassay.The resultsareshown inTable3,

andthemeanof the triplicatemeasurementisplotted

inFig.5.8-Ecells produced measurable quantities of hemagglutinin only atthe highest concentration of

virus used, whereas A-9 cellsproduced

hemaggluti-nin atallconcentrations of input virus used.

Sym-bols: A-9 (a);8-E (0).

cells, again there is abreak in the saturation curve at between 5 x 105 and 7 x 105 viral

particles boundper cell, reflecting the number

of specific binding sites. The nonpermissive

L1210 line shows little appreciable binding in

the concentration range used in this

experi-ment. Much lower levels of "specific" binding

mayexistfor theL1210cell line, but it is inno

waycomparable tothe A-9 system. The L1210 saturationdata whenplotted on alower scale

1 2 3 4 5 6

I.M.x 10-6

FIG. 5. ComparisonofMVMbinding to-control

A-9cells andtoresistant cloned derivativesofA-9cells

designated

8-E;

2 x104A-9or8-E cellssuspendedin PBS at

40C

werereacted with the indicated

multiplic-ityof[3H]thymidine-labeled MVMparticlespercell (inputmultiplicity [IM.])for2h.Sampleswerethen filtered, and the cell-associated radioactivity was measured and converted to bound multiplicity

(B.M.)asdescribed in thetext.Atall concentrations

ofinput virus,filtration of cell-free controlsamples

resulted in retentionofless than0.1% ofthe input

radioactivity, whereassampleswith cellsretained up

to75%.Thecontrol A-9 cells bind virusinabiphasic

manner, with saturation ofthefirstcomponent oc-curringatabout5 x105MVMparticlespercelland the second componentnotsaturable under these con-ditions. Symbols:A-9 (0); 8-E (a).

(one log unit) generate a monophasic curve similar to the 8-E curve (not shown). This indi-cates

that

the L1210 cells also bind virus with nonsaturating kinetics. The reaction is, how-ever, of even lower affinity than observed for virus binding by 8-E cells.

In view

of the

reportsthat

specific

tissuesare

susceptible

to

parvovirus

infection

during

the course

of

development (3, 12),

it was of interest VOL. 24, 1977

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in 0

2

1 2 3 4 5 6

I.M. x 106

FIG. 6. Comparison ofMVM binding to

permis-sive A-9 cells andtononpermissiveL-1210 cells: 2 x

105 cells (A-9 orL-1210) suspended inPBSat40C

were reacted with the indicated number of [3H]thymidine-labeledMVMparticlespercell(input multiplicity [IM.]) for2 h.Samples were then fil-tered,and thecell-associatedradioactivity was

mea-sured and convertedtoboundmultiplicity (B.M.)as

described in thetext.Symbols:A-9 (0);L-1210 (0).

Each point is the average ofthree determinations

plusorminus therange.

to

determine

ifthe number of viral

attachment

sites

changed

in cells

undergoing

"differentia-tion." Friend-745

erythroleukemia

cells canbe

induced to differentiate and produce hemoglo-bin in culture with Me2SO

(6).

These cellsare

permissive for MVM

growth

inuninduced cul-tures (16a). Friend cells appear to be a stem

cell in the erythropoietic series, which

ul-timately leads

in vivo to formation ofmouse

erythrocytes.

These

erythrocytes bind and

are

agglutinated by

the virus. In view

of these

facts,

we

wished

to determine if the

num-ber

of virus-binding

sites per cell changes

during

the induceddifferentiation of these cells

in

culture.

A

culture of uninduced Friend cells

was

split

into two

equal volumes

of

fresh

me-dia,

oneofwhich received 1.8%Me2SO.

Induc-tionwasmeasuredby counting the percentage

ofbenzidine

(hemoglobin)-positive

cells in each culture.

Initially

both cultures wereless than

1%

benzidine

positive, and the uninduced

cul-ture

remained

as

such

throughout successive

days.

The induced culture

steadily

increased in

the

proportion

of benzidine-positive

cells,

reaching

a

peak of

50% on day 4. Cells were

harvested and

suspended

in

cold

PBS,

and the

virus-binding assay as described for A-9 cells

above (see also Materials and

Methods)

was

used to titrate the number of specific binding sitespercell inthe

parallel

cultures.

Figure

7

shows that both induced and

uninduced

cul-tures possess

about

1.5 x 105

saturable binding

sites per cell,

and no difference between the two

is

detectable with our methods.

Electron

microscopy. In an attempt to

visu-alize the virus-binding sites on the surface of

the cell, A-9 cells in monolayer

were

exposed

to

106 virus

particles per cell

in

PBS at

4VC for 2 h.

The monolayers were rinsed and prepared for

electron microscopy as described in Materials

and Methods. Due to virus adsorption to the

substrate

(unpublished observations) as well as

equilibrium considerations (see

Fig. 5 and 6),

fewer than

5 x

105

particles are bound per cell

under these conditions. MVM can be seen to

bind to at

least three morphologically distinct

regions of the surface (Fig. 8 and 9). First of

all, clusters of virus,

as

well

as

single particles,

can

be

seen

on

the surfaces of numerous

filipo-dia. Small

patches of particles, as well as single

particles,

can

also be

seen

scattered

over

the

apparently unspecialized regions of the cell

sur-face. In

addition,

virus

particles can

be

seen

localized

in

specialized clefts

in

the cell

mem-brane. These clefts are characterized by a

prom-inent submembranous

thickening

and outer

surface glycocalyx and

appear to be

endocy-totic regions

of the cell surface (1, 7).

5-4

-0

I 2 3 4 5 6

I.MX 10 6

FIG. 7. Comparison of saturation binding

of

MVMtoinduced anduninduced Friend-745

erythro-leukemiccells;2 x 105inducedoruninduced

Friend-745 cells (Fig. 8)

suspended

inPBS at40C were reacted with the indicated numberof

tHithymidine-labeledparticles (inputmultiplicity [I.M.])

for

2 h.

Sampleswerethenfiltered,and thebound

multiplic-ity (B.M.) was measured as described in the text. Both induced and uninduced cultures bind virus withabiphasic curve, which goes

through

a

transi-tion at approximately1.5 x 105saturable

binding

sitesper cell. Symbols: uninduced Friend-745 cells

(0);induced Friend-745 cells (-).Each

point

is the average ofthree determinations plus or minus the range.

on November 10, 2019 by guest

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[image:6.504.68.258.59.240.2] [image:6.504.275.465.362.526.2]
(7)

VOL.24,1977~~~~~BINDINGSITES FOR MVM

217

Is,~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~sy

8

a~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~a

-A>~~~~~~~~A

.4.

wt~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~'

4~~~~~~~

46%~~~~~~~

e~~~~~~

b

~~~~~~~~~~~A*~~~~~~~~0

.-Jt

C

., Q 6

4.0-Z... '. I-A

)e

454'

*'~~~~~~~~~~~~~~407`

nA ~ ~ ~ ~

'~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~4

FIG. 8. Electron micrographs ofthe surface ofA-9 cells fixed-after a2-h exposure to 106JiOS MVM

particlespercell at4'C. (a) Low magnification ofA-9 cellshowingseveral scatteredpatches ofadsorbed MVM. Thearrows indicateafewsuchpatches. The crossedarrowspointoutclustersofvirionsadheringto

filipodia. x 18,000. (b) High-magnification micrograph showingalarge patch ofMVMboundto the cell

surface. The sectionwasapparentlycutperpendiculartotheplane ofthe cell membraneattheregionofthe viruspatch.Thisareaofthe membraneappears toberelatively unspecializedmorphologically.Anadditional

patch ofviruscanbeseentocontinuefromtheflatcellsurfaceupthe stalkofafilipodium(arrow) x60,000.

(c) Electronmicrograph showingvirusboundtofilipodia(arrow)as wellasatangentialsection througha

viru8 patchillustratingtheparacrystalinnatureofsuchpatchesatthe cellsurface(crossedarrow). x45,000.

rt.N

V. I'

4".

VOL.

24,

1977

on November 10, 2019 by guest

http://jvi.asm.org/

[image:7.504.46.447.81.595.2]
(8)

218

LINSER, BRUNING, AND ARMENTROUT

9a

41

_Nt

j~

c

;

,

.t

4$ r b~ .rs . X * * , a

.N

V r f

4;

A-i.

^

.- 4

A?

;IV t

log*~ ~ ~

eJ

Ar-8

Nt;- s~* .w

h-.40 V- .. 9%.^b- 4.0

~~~'tW

tv'Nsm

*Air~~~I

*r, fA s>

rz",T ~ .

i

It *fe~

* ; t*

C

V

I~N

0 : * C l:W~r4 1 *. a

;NR i i .~ S X

--SpsF;

FiG. 9. Electronmicrographs of the surface ofA-9cellspreparedasinFig. 8. (a) Micrographshowing

virusboundto unspecializedregions of surface membraneas wellas toa morphologicallydistinguishable

region ofmembrane. Thisspecializedregion ischaracterized byaprominentthickeningonthe inside(arrow

heads) and a less clearly visible glycocalyx on the outside surface of the plasmalemma. x60,000. (b)

Micrograph again showing virus particles adsorbed to a specific membrane region characterized by a

prominent submenbranous thickening(arrowheads) and lightly visibleouterglycocalyx (arrow). x65,000.

(c)MVMparticlesadsorbedtounspecialized regions of cell surfaceingroups(arrow)andassingleparticles

(arrow heads). x60,OOO.

,sk

.

rf,

J. VIROL.

v

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[image:8.504.68.466.75.588.2]
(9)

BINDING SITES FOR MVM 219

These

results demonstrate

that

when the

viral

receptor

sites are in excess,

virus is

ob-served

on

several regions of the cell surface.

Further

studies are in progress to delineate the

processes by which the bound virus enters the

cell.

DISCUSSION

Utilizing

a

radiolabeled virus

probe,

we

have

characterized and quantified the number of

"specific"

MVM

binding sites

on

the surface

of

several murine cell lines. The A-9 derivative of

mouse

L-cells

(13)

binds

virus

with

a

biphasic

saturation

curve

and

a

pH optimum of 7.2

at

4VC.

The

biphasic

saturation

curve can

be

bro-ken into a

rapidly saturable and

an

apparently

insaturable reaction.

There

are

approximately

5 x 10, saturable binding sites on A-9 cells.

Friend-745

erythroleukemic cells, another

mu-rine

cell

line

permissive for

MVM

growth

(16a),

also bind virus in a biphasic reaction

with

approximately

1.5 x 105

saturable

bind-ing sites per cell. The mouse

lymphoid cell line

L1210, however, is not

susceptible to MVM

infection and binds little

virus. A cloned

variant of the A-9 cell line

selected for

re-sistance to infection

with MVM

was also

de-veloped and shown to

apparently lack the

saturable component of the biphasic binding

curve.

These results indicate that the

satur-able

and,

consequently, specific binding

sites

on

A-9

cells

are

directly

involved in the

infec-tious process.

Most

of the cell-associated

counts

following

a

binding

reaction

at

4VC

are at

the surface

of the

cell and

not

internalized.

About

80%

of the

cell-associated

counts can

be washed off the cell

with

a

brief exposure

to

Ca2+,Mg2+-free

PBS

containing

0.001 M

EDTA.

After

a

2-h

incuba-tion

with

subsaturating

quantities of labeled

virus, at least

45%

of the

counts

can

be

removed

from

the cell

in a

1-h

chase

with excess

cold

virus.

This last observation suggests that the

binding

of MVM

at

4VC is

reversible

and will

consequently approach equilibrium.

Rough

estimates

of the initial

rate

of

attach-ment

were

calculated from the

slope

of

the

binding

curves

(K)

at 30

s,

as

described

by

Lonberg-Holm

and

Whiteley

(16). At

4VC

the

value equals 2.8

x 10-

7cmVmin,

and at

210

C K

equals

4.4 x 10-

cm:/min. This

represents

an

increase

in K

by

a

factor of

1.33/100C.

This

is

very close

to

the

predicted effects of

tempera-ture on the diffusion coefficient for

particles

this size

(i.e.,

1.30/100C)

(16, 22).

Thus, the

difference seen between reactions

occurring at

4

and

21'C seem to

be due

primarily

to

the

change

in

particle diffusion

rates.

Also,

since

the

phase

transition

for

membrane

lipids occurs

at

18'C (K.

Lonberg-Holm

and L.

Phillipson,

in

Tiffany and Blough, ed., Cell Membranes and

Viral Envelopes, in press), it does not

appear

that

the binding of MVM as measured here

re-quires free

lateral movement of receptors,

contrary to what has been reported for

adeno-virus

(17).

The

binding of

several different

picornavi-ruses, as

well as adenovirus, is affected

some-what more

by temperature than MVM

binding

(16),

although there

is

considerable

variability

(2, 11). The

initial

rates

of

attachment

for MVM

calculated

at

4

and

21°C

are

considerably

more

rapid than for

picornaviruses

in

general (i.e.,

10-8 to 10-9

cm:'/min

at 30 to

37°C)

(16).

The

theoretical

maximum

rate

of

1.7 x 10-7 cm

3/

min for

picornaviruses (16) is

also

somewhat

slower than

observed for

MVM.

This may

re-flect the

crudeness of

our rate measurements.

On the

other

hand,

this may

be

due

at

least in

part to

the

considerable

differences

that

exist

between the

picornavirus-receptor

relationship

and

MVM-receptor interactions.

Picornavi-ruses

bind to

104

receptors

per

cell,

whereas

MVM

receptors appear to be

about

50

times

as

numerous,

which

would

increase

the

rate

of

parvovirus

binding by

increasing

the number of

effective cell-virus collisions. The picornavirus

receptor reaction has been generally reported

as

being largely

irreversible.

MVM

binding

measured at 4°C is, however, readily reversible

and

apparently defined by second-order

equilib-rium

kinetics.

The

virus

preparations used to measure the

above

findings

were

well suited for these

pur-poses.

The

virus

was

radiolabeled

with

PH

thymidine

to

avoid such

changes

in

the

surface properties of the virus as might be

pro-duced by techniques such as radiolabeling with

iodine. The virus is isolated in velocity

gra-dients

to assure

that the particles are

monodis-perse

when

introduced into a reaction, an

im-portant consideration in view of the tendency

for

MVM

to

aggregate

when

placed into a

high-salt environment such as CsCl equilibrium

density

gradients. Once the virus has been

placed

into

physiological salt concentrations as

in

the binding reaction mixtures, it is

impossi-ble

to

control aggregation, and this may

con-tribute

to the

nonspecific

component of the

binding

reaction.

The

purity of the virus

probe was confirmed

by sodium dodecyl

sulfate-discontinuous gel

electrophoresis (18). Only viral proteins are

ob-served

in

the 110S material from the

sucrose

velocity gradients. All of the

radioactivity is

acid

precipitable and resistant to DNase. These

observations

indicate that

all of the the

cell-associated

radioactivity after a binding

reac-VOL. 24, 1977

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http://jvi.asm.org/

(10)

220

tion

indeed represents

adsorbed

or

internalized

virus.

The results

of the binding

of virus to

induced

and uninduced Friend-745 cells indicate that

the

virus-binding

sites do not

change

on

the

surface, although during

Me2SO-induced

differ-entiation the cell surface

undergoes several

al-terations

with the appearance

of

new

antigens

(8).

Whereas the viral attachment sites

are

present in

the extreme

form

of the

differen-tiated cell in vivo,

the

mouse

erythrocyte,

our

results indicate

that

virus-binding

sites are

probably

retained throughout the

differentia-tion process in

about the

same

numbers

per

cell

as present on

the

precursor

cells.

Furthermore,

in

light

of these

results

it

is

unlikely that the

uninduced cultures

of Friend-745 cells support

growth of

MVM

due

to

the presence of

a

few

spontaneously induced

cells that

have

viral

re-ceptors among a

large

population

of

resistant

cells lacking receptors.

It is worth noting

that

an

erythroid

tumor

line possesses virus-binding sites whereas the

lymphoid

tumor

line

(L1210)

appears

to

lack

receptors.

The difference between

lymphoid

and

erythroid

cells in

terms

of MVM infection

susceptibility has

been

previously

noted

by

Miller et al. (16a).

Preliminary

electron

microscopic

examina-tion

of

virus

bound

to

A-9

cells in

subsaturating

quantities

reveals

binding

to

several

morpho-logically distinct regions

of the cell surface.

A

comprehensive study

utilizing

specific

virus

la-beling

is

under

way

to

shed

further

light

on

the

nature

of the

specific, infection-related

binding

sites.

The

data we have presented on the binding of

MVM to

cells

can

be

explained by

a

simple

model: cells sensitive

to

viral infection bind

virus to

specific

sites

on

the

surfaces;

some

virus is

also

bound

nonspecifically,

but

such

binding

is very

inefficient

in

causing infection

of the cell.

However,

it

should be noted

that

our

viral

probe

consists

of the

full virus

class

of

particles, which

is

not

homogeneous.

The

110S

virus consists of at least two classes of

particles:

a

low but variable

percentage (5

to

25%) of

a

dense

(1.46

g/cm3

in

CsCl) precursor particle

and

a

high

percentage of

a

lighter

(1.42

g/cm3

in

CsCl) product particle

(4, 18). Some evidence

has been presented which might indicate that

the

minor, dense

species has

a

poor

affinity

for

cells

compared

with the

binding

of the

major

species (4).

In our

experiments,

we

have

ob-served

up

to 60 to 65% of

the

input

particles

bound

to

cells

when sites are in excess.

These

results would

support

the

idea

that our

binding

assay

measures the

binding

of the

predominant

1.42-g/cm3 density class particles. However,

re-cent results indicate that both classes of viral

particles are equally rapidly bound to cells

un-der our assay conditions (P. Linser and R. W.

Armentrout, in D. C. Ward and P.

Tattersall,

ed., Parvoviruses, in press). The fact

that the

viral preparations used in the binding assay

are not

homogeneous limits the

conclusions

that can be derived from the kinetic data.

Nevertheless, the heterogeneity of the viral

particle preparations does not affect our

conclu-sion

that specific cell surface receptors are

re-quired for efficient infection of cells.

ACKNOWLEDGMENTS

Wegratefullyacknowledge helpfuldiscussions with Pe-terTattersall and David Ward duringthe courseof this work. R. Morrisprovidedexperttechnical assistance.

This investigation wassupported byPublic Health Serv-ice grants 1 K04CA 00134and5R01 CA-16517awardedby the National CancerInstitute, grant 1-396from the Na-tional Science Foundation-March ofDimes, and a grant from theUnitedFundHealth FoundationofCanton,Ohio.

LITERATURE CITED

1. Anderson, R. G. W., J. L. Goldstein, and M. S. Brown. 1976.Localizationof lowdensitylipoprotein receptors on plasma membrane of normal human fibroblasts andtheirabsence in cells from afamilial hypercho-lesterolemia homozygote. Proc. Natl. Acad. Sci. U.S.A. 73:2434-2438.

2. Bachtold, J. G., H. C. Bubel, and L. P.Gebhart. 1957. The primary interaction of Poliomyelitis virus with host cells of tissue culture origin. Virology 4:582-589. 3. Bates, R. C., andJ. Storz.1973. Hostcellrangeand growth characteristics of bovine parvoviruses. Infect. Immun. 7:398-402.

4. Clinton, G. M.,and M. Hyashi. 1976.Theparvovirus MVM: a comparison of heavyand light particle infec-tivityand their density conversion in vitro. Virology 74:57-63.

5. DeBernardo, S., M. Weigele, V. Toome, K.Manhart, andW.Leimgruber.1974.Studiesonthe reaction of fluorescaminewith primary amines. Arch. Biochem. Biophys. 163:390-399.

6. Friend, C., W. Scher, J. G. Holland, and T. Sato. 1971. Hemoglobin synthesis in murine virus-induced leu-kemic cells in vitro: stimulation of erythroid differen-tiationby dimethylsulfoxide. Proc.Natl.Acad. Sci. U.S.A.68:378-382.

7. Friend, D. S., and M. G. Farquhar. 1967. Functions of coated vesiclesduringprotein absorptionintherat vas deferens. J. Cell Biol. 35:357-376.

8. Fujihami, N., Y. Sujimoto, and A. Hajiwara. 1973. Induction of erythroid maturation by DMSO in Friend leukemia cells. Dev. Growth Differ. 15:141.

9. Gierthy,J.F., K. A.0. Ellem,and I. I.Singer.1974.

EnvironmentalpH and the recovery of H-1 parvovi-rus during single cycle infection. Virology60:548-557. 10. Holland, J. J., and B. H. Hoyer. 1962. Earlystages of

enterovirus infection. Cold Spring Harbor Symp. Quant. Biol. 27:101-112.

11. Holland, J. J., and L. C.Mclaren.1959. The mamma-lian cell-virus relationship. II.Adsorption, reception andeclipseofparvovirusby HeLa cells.J.Exp. Med. 109:487-504.

VIROL.

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BINDING SITES FOR MVM 221 12. Kilham, L., and G. Margolis. 1970. Pathogenicity of

minute virusof mice for rats,mice, and hamsters. Proc. Soc. Exp. Biol. Med. 133:1447-1452.

13. Littlefield,J. W. 1964.Threedegrees of guanylic acid-inosinicacidpyrophosphorylase deficiency inmouse

fibroblasts. Nature (London) 203:1142-1144.

14. Lonberg-Holm, K., R. L.Crowell, and L.Phillipson.

1976.Unrelated animal viruses sharereceptors.

Na-ture(London) 259:679-681.

15. Lonberg-Holm,K.,and B. D. Korant. 1972.Early in-teraction ofrhinoviruses with host cells. J. Virol. 9:29-40.

16. Lonberg-Holm,K.,and N. M.Whiteley. 1976.Physical

andmetabolic requirements for early interactions of poliovirus and human rhinovirus with HeLa cells. J. Virol. 19:857-870.

16a. Miller, R. A., D. C. Ward,and F. H. Ruddle. 1977. Embryonal carcinoma cells (and their somatic hy-brids)areresistanttoinfectionby murine parvovirus MVM which does infect other teratocarcinoma-derived celllines. J. Cell.Physiol. 91:393-401. 17. Phillipson, L., E. Everitt, andK. Lonberg-Holm. 1976.

Molecularaspectsof virus-receptor interactioninthe

adenovirus system. In R. F. Beers, Jr., andE. G. Bassett(ed.), Cell membranereceptors for viruses, antigens andantibodies, polypeptide hormones,and smallmolecules. RavenPress, NewYork.

18. Richards,R., P. Linser, and R. W. Armentrout. 1977. The kinetics ofassembly of aparvovirus, minute

virus of mice, insynchronizedratbrain cells. J. Vi-rol. 22:778-793.

19. Rose,J. A. 1974. Parvovirusreproduction,p.1-61. In H. Fraenkel-Conrat and R. R. Wagner (ed.), Compre-hensivevirology,vol. 3.PlenumPress,NewYork. 20. Tattersall, P.1972.Replication of theparvovirusMVM.

I.Dependence of virus multiplication and plaque

for-mationoncell growth. J. Virol. 10:586-590. 21. Tattersall, P.,P. J.Cawte, A.J. Shatkin,and D. C.

Ward. 1976. Three structural polypeptides coded for by minute virus of mice, a parvovirus. J. Virol.

20:273-289.

22. Valentine, R. C., and A. C. Allison. 1959. Virus particle adsorption. I.Theoryofadsorptionandexperiments

onthe attachment ofparticlestonon-biological

sur-faces. Biochim.Biophys. Acta34:10-23. VOL. 24, 1977

on November 10, 2019 by guest

http://jvi.asm.org/

Figure

FIG.Minutes2filtrationMVM[3H]thymidine-labeledated4CC;cellscleporetime.the h1. Effect of temperature on the binding of to A-9 cells in suspension
FIG. 3.pointfor[3H]thymidine-labeledtionsdetermined.minusthex 105 pH optimum for MVM binding reaction; 2 A-9 cells suspended in various buffer combina-(listed in Table 2)were reacted with 105 viral particles per cell at 40C 2 h
TABLE 3. Hemagglutination activity produced in a tissue culture infectivity assay run on A-9 and 8-E cells inparallels
FIG. 6.plusEachdescribed105[3H]thymidine-labeledsuredsiveweremultiplicitytered, Comparison of MVM binding to permis- A-9 cells and to nonpermissive L-1210 cells: 2 x cells (A-9 or L-1210) suspended in PBS at 40Creactedwiththeindicatednumberof MVM particles
+3

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